JP6730308B2 - Laminated tissue graft products - Google Patents

Description

FIELD The present invention relates to tissue graft products useful in promoting regrowth and healing of damaged or diseased tissue structures. More particularly, the present invention is directed to laminated implant products formed from multiple sheets of biological and/or synthetic materials, and methods of making the products.

BACKGROUND Compositions of decellularized tissue from warm-blooded vertebrates, including humans, can be used as tissue graft material. Typical tissue graft compositions can be derived from the dermis, small intestine, bladder, renal capsule, single glandular stomach, and forestomach matrix (eg, the entire contents of which are incorporated herein by reference, US (See Patents 4,902,508, 5,554,389, 6,099,567, 7,087,089, and 8,415,159). Known as the extracellular matrix (ECM), these compositions have an important role in providing the optimal chemical and structural environment for tissue growth and regeneration. ECM scaffolds used for tissue regeneration are conventionally prepared from various organs, as well as decellularized human and animal tissues isolated from various animal connective and basement membrane sources. These scaffolds promote tissue regeneration and are immunologically well tolerated.

An ideal tissue graft is a piece of analog that is as close as possible to natural tissue. Tissue processing is required to remove cellular elements that can cause rejection and ensure safety from infectious diseases. Further processing may be introduced to customize the manufacture of the implant to meet site-specific needs and improve shelf life. Each successive processing step can damage the ECM, alter the immune response and consequently affect the tissue remodeling process. Chemical treatment, drying, and sterilization techniques damage the ECM and thus affect the in vivo behavior of implants (1-3). Ready-to-use products are preferred by surgeons. As a result, minimally treated wet implants are preferred.

One limitation of some ECM graft materials is that the thickness of the graft is determined by the thickness of the tissue layer obtained from the raw material. For example, the thickness of the forestomach ECM sheet is limited by the thickness of the original tissue. However, applications such as hernia repair, skin grafts, dura replacement, tendon repair and reconstructive surgery often result in thicker grafts providing adequate tensile strength, biomechanical performance, and biological activity. Need that.

Individual sheets of ECM tissue typically have anisotropic mechanical properties that are directionally specified by the orientation of collagen fibers within the tissue. This results in directional variability in the physical properties of a single natural sheet of ECM tissue. Laminated implant fabrications with alternating sheets with different fiber orientations can minimize fabrication directional variability and can result in strong isotropic fabrications in multiple directions.

Multiple sheets of ECM can be prepared by using techniques such as compression and drying, chemical crosslinking, suturing, or by using adhesives to customize the ECM implant to meet site-specific needs. It is possible to produce laminated implant products that include (see US Pat. Nos. 5,885,619, 5,955,110, and 8,415,159).

Implant products made using these techniques have limitations. When air, heat, or lyophilization is used to dry the implant product, the ECM is damaged as a result of water loss. In the case of lyophilization, ice crystal formation leads to structural changes in the scaffold (4). While dehydration and compression are used as a method of making laminated implant products, ECM proteins are typically damaged in processing, resulting in a hyperimmunological response. A further limitation of dehydrated and compressed products is their tendency to delaminate after rehydration, during surgical handling, implantation and over time thereafter.

Sutures may be used to overcome the tendency of delaminated implant products to delaminate.
However, sutures can introduce foreign material into the implant, resulting in inflammation, scarring, and encapsulation, which may be undesirable in some situations (5). For example, manufacturing a laminated ECM implant product using permanent synthetic sutures, such as Prolene, to secure the sheets to each other results in ECM replacement over time, but synthetic sutures are remodeled. Not done. This is undesirable in applications where the implant must completely replace the patient's own tissue. Permanent sutures pose a high potential for infection, limiting the usefulness of this type of product in open dermal repair applications. Implant products containing absorbable sutures have a limited shelf life in the moist location (6, 7) because sutures are often hydrolytically disintegrated. As a result, absorbable sutures are not suitable for applications that require high tensile strength for extended periods of time.

Laminated implant products may also consist of ECM sheets held together using adhesives. However, the introduction of adhesives may create a barrier to cell migration and change the typical mechanism and kinetics of ECM remodeling.

Implant products comprising chemically cross-linked sheets of ECM bonded to each other are known to induce foreign body reactions and have limited bionutrient properties.

Implant products consisting only of synthetic polymer mesh are known to provide long-term strength and stiffness. However, over time, large amounts of synthetic material can cause foreign body reactions, which can result in mesh erosion that allows the mesh to pass through layers of tissue. Synthetic polymeric material implants have no biological components and therefore do not provide biological support for wound and tissue repair. This can lead to encapsulation of the implant and increase the risk of adverse reactions.

Thus, including ECM and/or natural or synthetic polymeric materials that can be easily tailored to meet the biophysical needs of a wide range of anatomical sites and do not delaminate during surgery or after implantation. The need for laminated implant products can be identified from the problems and drawbacks associated with existing implant products. Further, implant products that remain intact in dry and moist locations are desirable. Most preferred is a laminated implant product that is presented in a moistened form to avoid damage to the ECM and to retain the inherent bionutrient properties.

Accordingly, it is an object of the present invention to provide a tissue implant product comprising two or more layers of ECM or polymeric material that at least partially overcomes one or more of the above mentioned problems, or a useful alternative. To at least provide existing products or operations.

SUMMARY The present invention does not use any synthetic materials such as sutures or adhesives that hold the layers together, thus minimizing the risk of rejection, inflammation, or unwanted encapsulation, extracellular matrix (ECM). ) Or a tissue implant product comprising two or more layers of polymeric material.

Accordingly, in a first aspect of the invention, a tissue implant product comprising two or more layers of material, each layer comprising an extracellular matrix (ECM) or polymeric material, the layers comprising: Mating one portion with another layer provides a tissue graft product that is laminated to each other.

In certain embodiments of the present invention, a tissue implant product comprises a first layer of material having a plurality of lugs and a second layer of material having a plurality of holes, each of the first layers being The lugs are placed through the holes in the second layer and the holes and lugs are dimensioned such that the lugs engage the surface of the second layer to engage the first layer with the second layer. Have.

The tissue implant product may have at least one layer containing ECM, or may have a layer of any material containing ECM. The product may include any suitable number of layers of ECM and/or polymeric material, eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 layers of ECM and/or polymeric material. Good. The ECM may be formed from the proprio-submucosa of the ruminant forestomach. The tissue implant product may have at least one layer that includes a polymeric material, or may have a layer of any material that includes a polymeric material. The polymeric material may be a synthetic material formed from polypropylene, polytetrafluoroethylene, polyglycolic acid, polylactic acid, polygrecapron-25, or polyester. Alternatively, the polymeric material may be a natural material such as a protein, polysaccharide, glycoprotein, proteoglycan, or glycosaminoglycan. Examples may include collagen, alginate, chitosan, and silk.

The holes in each of the second layers may have any suitable shape, including substantially circular, triangular, square, rectangular, diamond-shaped, or star-shaped. Circular holes typically have diameters in the range of 2-4 mm. Each layer having a plurality of pores may have a density of pores of 0.5 to 15 pores per cm ² .

The tissue graft product of the present invention may be a substantially flat sheet or may have a three dimensional form shaped to fit where the product is to be implanted.

The tissue graft product may be moist or dried, for example by freeze-drying.

In a second aspect, the invention is a method of preparing a tissue graft product of the invention comprising laminating two or more layers of material, each layer comprising an extracellular matrix (ECM) or Provided is a method comprising a polymeric material, wherein layers are laminated to each other by interlocking a portion of one layer with a portion of another layer.

In some embodiments of the invention, the method comprises
(I) applying a first layer of material having a plurality of lugs to a second layer of material having a plurality of holes;
(Ii) pushing the lugs of the first layer through the holes of the second layer, the holes and the lugs engaging the lugs with the surface of the second layer to bring the first layer into the second layer. A process having dimensions such that it meshes with,
including.

In another aspect of the invention,
(I) a mold on which sheets of ECM or polymeric material can be stacked,
(Ii) lug cutting means for cutting lugs into a sheet of ECM or polymeric material to form a rug sheet.
(Iii) perforating means for creating perforations in a sheet of ECM or polymeric material to form a perforated sheet;
(Iv) means for pushing the lugs of the lug sheet through the perforations of the perforated sheet to form a tissue implant product;
There is provided a tissue graft product of the present invention comprising:

In a further aspect of the invention there is provided the use of the implant product of the invention for replacing or repairing human or other animal tissue.

1 is a schematic representation of a tissue graft product of the present invention. 1 is a cross-sectional view of a schematic representation of a tissue graft product of the present invention. 1 is a cross-sectional view of a schematic representation of a tissue graft product of the present invention. 1 is a schematic representation of an example of the device used to prepare the tissue graft product of the present invention. 3 illustrates a process for preparing a tissue graft product of the present invention. 1 is a schematic representation of an example of the device used to prepare the three-dimensional tissue implant product of the present invention. 3 is a bar graph showing wet handling integrity of lugged and unlugged sheep forestomach matrix. 3 is a bar graph showing wet handling integrity of rugged and unrugged sheep rumen matrix. 3 is a bar graph showing wet handling integrity of lugged and unlugged sheep pericardial matrix.

DETAILED DESCRIPTION OF THE INVENTION Definitions The term "extracellular matrix" (ECM), as used herein, refers to decellularized animal or human tissue, which carries the matrix and other materials for structural integrity. To provide a frame body.

The term "decellularized" as used herein refers to the removal of cells and associated debris from a portion of a tissue or organ, such as from the ECM.

As used herein, the term "polymeric material" refers to large molecules or macromolecules containing many repeating subunits, including, but not limited to, polypeptides and proteins (eg collagen), polysaccharides ( Natural materials including, but not limited to, polypropylene, polytetrafluoroethylene, polyglycolic acid, polylactic acid, and polyesters, including, for example, alginates) and other biopolymers such as glycoproteins. It may be a synthetic material containing.

As used herein, the term "mating" or "mating" refers to the engagement and mating of two or more sheets of overlapping material.

As used herein, the term "laminate" or "laminate" refers to the superposition of a sheet of material on a sheet of another material.

The term "sheet" as used herein refers to a substantially flat flexible portion of ECM or polymeric material.

As used herein, the term "layer" refers to two or more sheets that are interdigitated with each other, either overlapping or adjacent to each other.

As used herein, the term "lug" refers to a portion of a partially cut sheet such that the lug remains fixedly attached to the sheet via a connecting bridge.

As used herein, the term "lug sheet" refers to a sheet having a plurality of lugs cut into it.

As used herein, the term "perforated sheet" refers to a sheet having a plurality of holes.

As used herein, the term "threading the lug" refers to the process of pushing the lugs of the rug sheet through the holes of the perforated sheet.

The term "lug-threaded sheet" as used herein refers to a rug sheet in which the lugs of the lug sheet are pushed through the holes of the perforated sheet.

Laminated Graft Product The present invention generally relates to the lamination of two or more sheets of ECM or polymeric material in which one sheet is held together by mating a portion of another sheet with another sheet. The present invention is not limited to any particular mating method, but the lugs of one or more sheets are pushed through holes in other sheets so that the sheets are mated and retained together to form a laminated implant product. Will be described. The laminated implant product of the present invention offers advantages over other types of laminated products, including wound repair and tissue regeneration, and requires additional tensile strength beyond that of a single sheet of ECM. Are useful in a variety of clinical and therapeutic applications, including applications such as hernia or ligament/tendon repair.

Some embodiments of the invention are made without the use of any additional materials, such as sutures or adhesives, which may adversely affect or render the device unsuitable for wound or tissue repair, or manufactured. Featuring sheets that are laminated without the use of a drying step in the process. These laminates have greater tensile strength than the individual sheets. These laminates are also perforated, facilitating fluid drainage and reducing the risk of seroma formation.

Some laminated products described in the prior art laminated without the addition of other compositions tend to delaminate on prolonged hydration, making them unsuitable for wet locations. The products of the invention do not delaminate after prolonged exposure to water and continue to maintain structural integrity.

In general, the invention is based on a method that includes stacking multiple sheets of ECM or polymeric material and interlocking portions of the sheets to form a laminated implant product.

Extracellular Matrix The ECM-derived matrix used in the present invention is a collagen-based biodegradable matrix containing highly conserved collagen, glycoproteins, proteoglycans, and glycosaminoglycans in their native composition and concentration. is there. One extracellular collagen matrix used in the present invention is the warm-blooded vertebrate ECM. ECM can be obtained from a variety of sources, for example, gastrointestinal tissue taken from animals raised for meat production, including pigs, cows, and sheep, or other warm-blooded vertebrates. Vertebrate ECM is a rich by-product of commercial meat production operations and is therefore a low cost tissue graft material.

ECM tissues suitable for use in forming implant products include naturally associated ECM proteins, glycoproteins, and other factors naturally found in ECMs based on the source of the ECM. One source of ECM tissue is warm-blooded vertebrate forestomach tissue.

Forestomach tissue is the preferred source of ECM tissue used in the present invention. Suitable forestomach ECMs typically include the rumen ruminant forestomach propria-submucosa. In certain embodiments of the invention, the propria-submucosa is derived from the rumen, reticulum, or rumen of the forestomach. These tissue scaffolds typically have a contoured luminal surface. In one embodiment, the ECM tissue scaffold may additionally include decellularized tissue that includes a portion of the epithelium, basement membrane, or muscle layer and combinations thereof. The tissue scaffold may also include one or more fibrous proteins including, but not limited to, type I collagen, type III collagen, or elastin, and combinations thereof. These sheets are known to vary in thickness and definition depending on the source of vertebrate species.

Proprietary-submucosal tissue typically has an abluminal and luminal surface. The luminal surface is the surface that faces the lumen of the organ source and the anti-luminal surface faces the smooth muscle tissue surface. Proprietary-Multiple sheets of submucosa contact the abluminal surface with the abluminal surface, the abluminal surface with the abluminal surface, or the abluminal surface with the abluminal surface of the adjacent sheet of the ECM. Can be contacted and superposed. All these combinations of overlapping sheets of ECM from several or different vertebrate or organ sources produce a laminated implant product that includes the ECM.

One method of preparing the ECM used in accordance with the present invention is described in US Pat. No. 8,415,159. A segment of the vertebrate forestomach, preferably from a sheep species, is subjected to transmural osmotic flow between both sides of the tissue such that the tissue layers within all or a portion of the tissue are separated and/or decellularized. Exposed. The transmural osmotic flow can be directed from the lumen of all or part of the tissue to the abluminal side or from the abluminal side of the tissue or all of the tissue to the abluminal side. This may be achieved by separating the tissue such that the transmural osmotic flow is from a hypotonic solution to a hypertonic solution, for example between a hypertonic solution and a hypotonic solution. The method may further involve the removal of all or part of the tissue layer including the epithelium, basement membrane, or muscle layer, and combinations thereof. The hypertonic and hypotonic solutions may include, for example, water and optionally at least one buffer, detergent, or salt. A hypertonic solution contains a higher concentration of solute than a hypotonic solution. In certain embodiments, the hypertonic solution comprises 4M NaCl and the hypotonic solution comprises 0.28% Triton X-200 and 0.1% EDTA. In another particular embodiment, the hypotonic solution comprises 0.1% SDS. In yet another embodiment, the hypotonic solution comprises 0.028% Triton X-20, 0.1% EDTA, and 0.1% SDS. ECM can be stored in a hydrated or dehydrated state. Freeze-dried or air-dried ECMs can be rehydrated or partially rehydrated and used in accordance with the invention without significantly compromising bionutrient and mechanical properties.

Polymeric Material In some embodiments of the invention, a sheet of polymeric material may be included in the product as a rug sheet or a perforated sheet. For example, additional strength or long-lasting properties are found in polypropylene (found in Prolene mesh and Elevate mesh), polytetrafluoroethylene (found in Gore-Tex mesh), polyglycolic acid (found in Vicryl mesh), ( Incorporated into products by including delicate woven permanent synthetic materials such as polylactic acid (found in Paritex progrip mesh), polygrecapron-25 (found in ltrapro mesh), and polyester (found in Mersilene mesh) May be. Synthetic materials such as polypropylene, PTFE, and polyester are non-absorbable and last indefinitely, providing long-term strength and rigidity. Synthetic materials such as polyglycolic acid, polylactic acid, and polygrecapron-25 are absorbable meshes that provide additional strength in the short term but are absorbed in the long term. Alternatively, the polymeric material may be natural or derived from natural materials such as proteins (eg collagen), polysaccharides (eg alginate), glycoproteins, or other materials.

In other embodiments, the product may include a sheet of polymeric material only (ie, no sheet of ECM).

General Method of Preparing an Implant Product In one embodiment of the invention, a laminated implant product is formed from multiple superposed or partially superposed sheets. The dimensions of the individual sheets used are not critical. One method of forming a laminated implant product of ECM and/or SPM involves forming a number of lugs throughout the sheet and pushing a suitably shaped cutter (lug cutter) through the sheet to form a "lug sheet". Including. The rug sheet may be a wet sheet, a dry sheet, a freeze-dried sheet, a rehydratable sheet, or a rehydrated sheet. The rug layer may include one or more combinations of rug sheets and may or may not overlap each other. The rug layer may also include different combinations of different patterns of lug sizes to improve the strength of the overall layers of the laminate. A "perforated sheet" is formed by piercing a sheet of ECM or polymeric material with a sharp or blunt needle to form a large number of small perforations throughout the sheet. The perforated sheet may be a wet sheet, a dry sheet, a freeze-dried sheet, a rehydratable sheet, or a rehydrated sheet. The perforated layer may include one or more combinations of perforated sheets and may or may not overlap each other. The lug layer is at least partially overlaid on the perforated layer. At this point in the method, the layer may be rehydrated with a liquid such as isotonic saline. The lugs of the lug layer are then pushed through the perforations of the perforation layer using pins, push rods, punches, blunt needles, or similar devices to engage and secure the lug layer to the perforation layer. This forms the laminated implant product of the present invention.

Referring to FIG. 1, a laminated implant product 1 is shown including a rug sheet 2 and three perforated sheets 3. Each part 4 shows where the lugs are cut from the rug sheet 2 and pushed through the perforations in the perforated sheet 3 to the underside of the implant product 1. A single lug 5 is drawn pushed from the lug sheet 2. Other lugs are not shown. FIG. 2A is a cross-sectional view of a laminated implant product. The rug sheet 2 is shown superimposed on the four perforated sheets 3. The lug 5 is shown pushed from the lug sheet 2 through the perforations of the perforated sheet 3. Each lug 5 remains attached to the lug seat 2 via a connecting bridge 6. In fact, it will be appreciated that each lug 5 lies substantially flat against the underside of the implant product 1. Therefore, all sheets are meshed and held together.

In some embodiments of the invention, a laminated implant product consists essentially of ECM tissue, without potentially dangerous sutures, adhesives, and chemical pretreatment, and is used to form the product. It has greater mechanical strength and collagen content per cm ² than individual sheets.

The amount of tissue overlapping between adjacent sheets will vary depending on the application, the desired properties, the required laminating strength, the required surface area or size of the product, but at least a portion of each sheet will differ from that of another sheet. Overlap and mesh. The lugs engage and secure the perforated sheet to the rug sheet in the area of overlap to provide a laminated implant product.

The term “mating” or “mating” as defined above refers to 1 without the need for the addition of other materials such as adhesives or sutures, or the need for treatments such as compression and dehydration. A method in which one or more rugged sheets secure one or more perforated sheets to one another. The formed products may differ in the number of layers and sheets that are superimposed and secured at different points in the laminated implant product. The variable structure of the implant product can provide high mechanical strength.

The interlocked sheet strength of the product is mainly determined by the surface area of the overlapping lug-threaded perforated sheets, the fixed density, the strength of the lug connecting bridges, the rigidity and resistance to lug compression, the strength of the perforated sheet holes, and Due to the tightness of the fit between the lug and the perforation. A layer of product with small perforations and large lugs is fixed with great strength, thus reducing the possibility of lug withdrawal. However, too small perforations impede simple lug threading (lug threading) and are susceptible to breakage due to tearing of the drilled holes.

In a preferred embodiment of the present invention, the rug sheet comprises lyophilized tissue and may be hydrated when superposed on the perforated sheet prior to pushing the lug through the perforated sheet. However, in other embodiments, the rug sheet has sufficient properties to resist tearing, perforations, and other deformations that are detrimental to the process of lug cutting and lug pushing, so that the lugs do not deform and lag behind the perforations. The rug sheet may include a wet sheet, a dry sheet, a rehydratable sheet, or a rehydrated sheet, or a combination thereof, if not otherwise removed.

In some embodiments of the invention, the perforated sheet comprises moist tissue. However, in other embodiments, the perforated sheet has sufficient properties to resist tearing or other deformation that is detrimental to perforation, lug pushing, and lug retention, as long as it is a dry sheet, lyophilized sheet, re-watered sheet. It may include a compatible sheet, or a rehydrated sheet, or a combination thereof.

Apparatus for Preparing an Implant Product In a typical method of preparing an implant product of the invention, the sheets used to prepare the lugs and perforated sheets of the invention are placed on a mold. In one embodiment, the sheet may be stretched in both length and width directions on the mold to create tension in the sheet to allow for effective lug formation, perforation, and/or lug pushing. The stretched sheet is pierced by sharp or blunt end pins around the periphery of the mold and placed over the pins to maintain the tension of the stretched sheet. Alternatively, the sheet may be stretched and placed on the mold and the tension in the sheet maintained using a stop or press, or other suitable method.

A guide, such as a rod, bar, or similar shaped device, passes through the stretched sheet to provide a method of securing the stretched tissue to the mold and aligning any subsequent sheets that are added. And can be passed into the mold. These guides can also be used to align instruments used in the process, including instruments used to create perforations and push lugs through. The use of a mold with such a guide ensures that the lugs of the rug sheet and the perforations of the perforated sheet are aligned above and below each other, and that the lug pushing device is also aligned over the drilled hole. And can be reliably guided through the drilled hole.

In some embodiments, the sheets are placed on a mold and pinched using a rod, bar, or similar shaped device that can pass through the unstretched/tensioned sheet. In some embodiments, the sheet is placed on a mold and secured using methods including, but not limited to, stops or presses.

The composition of the mold is not critical and can be designed in any size or shape. In one embodiment, the mold is a 16 mm thick acetal block. The mold includes bosses for lugs, needles, and pins to pass into the mold. The density, shape, size, orientation, and mold of the array of fool holes, and the size, orientation, shape, and mold of the fool holes themselves are tailored to the type, thickness, and number of sheets and layers of material, and the desired application. Can be adjusted. In one embodiment, the fool holes are arranged in horizontally and vertically aligned rows and have a diameter of 3.1 mm and are spaced 3.5 mm apart at the centers.

In another embodiment, the fool holes are offset in horizontal or vertical rows to allow for reduction of the force required for cutting the lugs and/or to allow for denser lugs and fixation points. Is located in. In another embodiment, the mold is made from a group of small diameter rods with the ends of the rods forming the mold surface. In such an embodiment, the rod tip moves to allow passage of lugs, needles, and/or pins. In another embodiment, the mold is made of foam that separates to allow passage of lugs, needles, and/or pins. In these embodiments, it is not necessary to align the mold with the processing tool.

In some embodiments, the mold has a flat, flat surface and is used to form a planar laminated implant product.

In a typical process, a sheet of ECM or polymeric material is placed on a mold and the lugs are cut with a cutter of suitable shape (lug cutter) to form the rug sheet. The rug sheet is removed and a new sheet of ECM or polymeric material is placed on the mold. Alternatively, a new sheet is placed on a different mold with similar fool holes and guide patterns. A new sheet is perforated using needle(s) to form a perforated sheet. Subsequently, one or more lug sheets are overlaid on one or more layers of perforated sheet, the lugs of the lug sheet being applied to the layer of perforated sheet using non-sharp (blunt end) pin(s). Pushed through. When the lug is pushed through the holes in the perforated sheet, the lug opens, thereby engaging the rug sheet and the perforated sheet together to form a laminated implant product. Implant products can be prepared with at least two sheets, but may include 3, 4, 5, 6, 7, 8, 9, 10 or more sheets.

When a wet sheet is used, a separating slip may be utilized between the sheet, rug sheet, perforated sheet and the instrument element. This reduces the surface tension that allows the instrument elements to be easily separated from the sheet and other instrument elements without exerting a significant force on the sheet. Separation slips are typically made of a thin, flexible material such as polytetrafluoroethylene (PTFE) or stainless steel, and are wound from the sheet for removal without significant force on the sheet. It can be peeled off or peeled off.

A retainer plate may be used to secure the tissue prior to lug cutting, perforating, and pressing the lug through the perforated sheet. In some embodiments, the lug cutter may be a knife made from a sharp-ended metal tube. In other embodiments, the lug cutter may be made of any material that can be sharpened to the blade. Rug cutters cut lugs into circular shapes, including but not limited to squares, rectangles, triangles, or inverted triangles, diamonds, stars, etc. that keep the connecting bridge attached to the sheet. It may be cut into other shapes.

In some embodiments, the lug is as small as possible so that the protruding free tip of the lug has the least penetration. However, the lug must have sufficient width to achieve retention. For example, lug sheets having a lug size of 2.8 mm diameter and spaced 3.5 mm in the center are preferred for the preparation of 5-sheet products including forestomach proprio-submucosal ECM sheets. The connecting bridge of each lug is preferably as narrow as practicable while the lug pushing operation is still ongoing. A 0.7 mm wide connecting bridge is ideal for a 5-sheet product containing forestomach proper-submucosal ECM. The small width of the lugs and connecting bridges may be suitable for use with rigid lug threading materials, for lamination of a small number of sheets, or for thin sheets. Large lugs and connecting bridge widths may be suitable for laminating a large number of sheets, thick sheets, flexible materials, or where the presence of large free lug tips is not a concern.

In some embodiments, the rug sheet is created by cutting the rug using a plurality of lug cutters arranged in a flat block. The pattern of the rug cutter will match the stupid holes on the mold. The lug cutting block is aligned with the guides and fool holes on the mold and pressure is applied to the block to cut the lugs into a fixed sheet on the mold to form the lug sheet. The biasing force for applying pressure can be generated by any number of suitable methods including hand force, weight application, electronic pressing, or hydraulic pressing. The rug cutting operation can be applied multiple times on a large sheet to create a large rug sheet. In other embodiments, the lug cutters may be used individually, arranged in a flexible sheet, attached to a robot arm, or the lugs include, but are not limited to, a die or a laser. It may be cut using other cutting instruments, such as

In some embodiments, the drilling operation may be performed using a sharpened sharpened needle with a long taper. An anterior gastric peculiar-submucosal tissue ECM sheet in which one sheet is a rug sheet having 2.8 mm wide lugs with a 3.5 mm center gap between the lugs and 0.7 mm wide connecting bridges For lamination of a 5 sheet product containing a., needles having a diameter of 1.2 mm, spaced 3.5 mm in the center, may be used to pierce the sheet to create holes. However, needles having a diameter of 1.8 mm or less and greater than 1.8 mm may produce a proper engagement in the finished product. Short needle diameters may be suitable for mating with narrow width lugs or when the lug threading material is flexible.

In some embodiments, the perforated sheet is created by perforating the sheet with a needle. Multiple needles may be arranged in a flat block. The pattern of needles will match the stupid holes on the mold. Similar to the application of force to cut a lug into a sheet and form a lug sheet, a force is applied to the needle block to make a small hole in the sheet.

Laminated products are formed by partially or completely overlapping and aligning one rug sheet on one or more layers of perforated sheets and performing a lug threading operation. In some embodiments, the one rug sheet is rugged over the one or more perforated sheets. Lugs and Perforations The lugs and perforations on the sheet can be aligned by any suitable method, including, but not limited to, by eye, using a guide, or using a jig. The mated laminated implant product is formed by pushing a lug through the perforations to secure the layers and sheets together.

In some embodiments, the lug threading operation is performed using one or more blunt, rounded, and/or non-sharp pins (push through pins). Pre-gastric propria-mucosa with 2.8 mm wide lugs centered 3.5 mm apart, one lug sheet with 0.7 mm wide connecting bridge and perforations made with 1.2 mm diameter needles For lamination of a 5 sheet product containing the underlying tissue ECM, the lugs may be pushed through using a 0.55 mm diameter push-through pin. However, push-through pins with diameters of less than 1 mm and greater than 1 mm produce a proper mesh.

In some embodiments, the push-through pins are arranged in a flat block in a pattern that matches the pattern of fool holes on the mold. The pin block is aligned with the guides and fool holes on the mold and a biasing force is applied to the block to push the lugs of the lug sheet into the fool holes through one or more perforated sheets to allow the pins to move. Leave the lug in this position after it is retracted from the seat and removed. This results in an implant product in which the pressed lug stops one or more perforated sheets on the rug sheet. The lug threading operation may be applied multiple times on a large laminate to create a large laminated sheet.

FIG. 3 is a schematic representation of an example of the device used to prepare the tissue graft product of the present invention. The mold 11 (dotted line) is shown overlaid with a sheet 12 of ECM or polymeric material attached to the mold 11 using mold peripheral pins 13 to maintain tension in the sheet 12. The pressure plate 14 and the separation slip 15 are shown sandwiched by the mold 11 with a stop 16. The piercing needle block 17 (alternatively it may be a lug cutter block, piercing needle block or push-through pin block) and the spacer block 18 may be guided onto the mold 11 using a guide rod 19. The needle 20 projects from the lower side and is held by the block 17. When downward pressure is applied to the piercing needle block 17, the needle 20 is pushed downward to pierce the sheet 12. The piercing needle block 17 may be replaced with a lug cutter block having a lug cutter protruding from the lower side. In a similar operation, when downward pressure is applied to the lug cutter block, the lug cutter is pushed downward to cut the lug into the sheet. Further, the piercing needle block 17 may instead be a push-through pin block having a push-through pin protruding from the lower side. When downward pressure is applied to the push-through pin block, the push-through pin is pushed downward to push the lug through the perforations in the sheet, resulting in the mated laminated implant product of the present invention.

FIG. 4 illustrates the process of preparing the tissue graft product of the present invention. Steps A and B show the preparation of rug sheets. Steps C and D show the preparation of perforated sheets. Steps E and F show the fabrication of a laminated implant product. The lug cutter block 21 holds a plurality of lug cutters 22. The ends of the lug cutter 22 are angled such that a semi-circular section 25 is made in the sheet 23 when pushed through the sheet 23 of ECM or polymeric material held on the bottom 24. The lug cutter block 21 moves up to remove the lug cutter 22 from the sheet 23, and is rotated 90 degrees and pushed downward to cut the sheet 23 again. This is repeated twice so that the three-step operation results in the cut lugs 26 to form the rug layer 27. Referring to step C, a perforated needle block 28 holding needles 29 is shown positioned on three sheets of ECM or SPM 30. The perforation needle block 28 is pushed downward so that a plurality of perforations 31 are made in the sheet 29 to form the perforation layer 32. The lug layer 27 is overlaid on the perforation layer 32. The push-through pin block 33, which holds the plurality of push-through pins 34, is configured so that the push-through pins 34 push the lugs 26 through the perforations 31 to engage the perforation layer 32 with the lug layer 27 to form a laminated implant product 35. Is pushed into.

Implant Products Comprising Multiple Sheets In some embodiments, the formed product may include multiple rug sheets. Sheets may be created that have lug pattern arrays that are offset to the lug patterns of other rug sheets. This avoids multiple lugs on different lug sheets arranged on top of each, and multiple lugs pushed through the lug holes of the overlaid lug sheets. These lug sheets can include perforations such that the lugs can be pushed through the lug sheet and into the perforated sheet. Sheets designed with rugs and perforations having different patterns are termed "differential rug/perforated sheets".

In another embodiment, a layer comprising one or more rug sheets and/or a differential rug/perforated sheet is placed above and below a layer of one or more perforated sheets, with one layer between the layers of the sheet containing the rug. The layers of one or more perforated sheets may be effectively sandwiched. The result is a thick and strong product. Sandwiched laminated implant products are formed by pushing lugs through the perforations to secure the layers and form a sandwich. The lugs of the upper lug layer are pushed through the perforations from the top and the lugs of the sheet of the lower lug layer are pushed through the perforations from the bottom. The lugs may or may not be pushed through all the perforations and may or may not be pushed through the opposing lug sheet or the differential lug/perforated sheet.

Referring to FIG. 2B, three perforated sheets 3 are shown sandwiched between an upper rug sheet 7 and a lower rug sheet 8. The lug 9 from the rug sheet 8 is pushed through the perforations of the perforated sheet 3, and the lug 10 from the rug sheet 7 is also pushed through the perforations of the perforated sheet 3.

In a sandwich-like product, the lugs of the upper and lower lug sheets and/or the differential lug/perforated sheet may be offset from each other so that the lugs from opposite sides do not come into contact. A sandwich-like product in which the lugs are not forced through all layers or sheets offers advantages in situations where exposed lugs can cause tissue wear and/or friction. Thus, a sandwich-like device is ideal when the lug sheet and/or the differential lug/perforated sheet comprises a synthetic material that has the potential to be rigid and rough to tissue.

Pseudo-Isotropic Graft Product In some embodiments, a pseudo-isotropic laminated implant product is prepared from multiple sheets of ECM. As used herein, the term "pseudoisotropic" refers to a tissue graft material that has similar physical properties along each axis of the graft material. The pseudo-isotropic laminated implant product of the present invention may be prepared from individual sheets of ECM. ECM materials are typically strong in one direction compared to the other. This is often due to the alignment of ECM polymer fibers (eg collagen). A method of preparing a pseudo-isotropic laminated implant fabrication comprises overlapping at least a portion of a first sheet of rug or perforated sheet with a second sheet of rug or perforated sheet, the length of the first sheet The second sheet rotates so that the directional axis is at a constant angle to the longitudinal axis of the second sheet. Additional sheets of lugs or perforated sheets may be added as well to create a pseudo-isotropic laminated implant fabrication with the desired number of laminated sheets.

Large Area Implant Products Large area implant products can be prepared according to the present invention. Since the ECM is obtained from the tissues of certain animal organs, there are limits to the size of the sheets of tissue that can be used for transplantation work. When a large area sheet of graft tissue is needed (eg, due to a large burn), sutures, adhesives, or other types of treatments are used to create a graft product with sufficient surface area. It must be. However, the mating of partially overlapping sheets of ECM according to the present invention allows for the preparation of laminated implant products having a surface area greater than the surface area of any individual sheet used to prepare the implant product. ..

Three-Dimensional Implant Products In some embodiments, the implant products are essentially flat flexible products, prepared using a flat flat mold. In other embodiments, the implant product may have a curved three-dimensional shape. The ability to use a curved mold to lug a sheet through a sheet to form a three-dimensional shape from the sheet, so that the sheet retains its shape without introducing other materials or using other processes, is an existing technology. Advantageously, it overcomes the limitations of and allows the creation of laminated products that match the natural shape of a part of the human body. Flat laminated implant products have limited ability to conform to natural curved shapes such as breast implants and are prone to groove or wrinkle formation. These wrinkles may be sites of seroma formation leading to complications or worsening of cosmetic effects. Therefore, the ability to form the device into a three-dimensional conformed shape during the mating process is advantageous.

A three-dimensional shaped interlocking laminated implant product may be formed using a mold having a curved shape. The mold may also have stepped edges or multiple flat surfaces arranged at different angles. The mold may have non-vertical fool holes for non-vertical lug cutters, needles, and pins.

The lugs and perforated sheets are preferably hydrated during the manufacturing process so that the tissue can easily fit into the preferred shape.

A mold with fool holes perpendicular to the mold surface may be used with individual lug cutters, flexible sheet rug cutters, rug cutters on robot arms, lasers, or other techniques. In another embodiment, the lug cutting block may be used with a mold having fool holes arranged non-perpendicular to the surface. Alternatively, the three-dimensional shape may be formed by a mold having multiple flat surfaces and a lug cutting block that conforms to the individual flat surfaces. Similarly, molds with fool holes perpendicular to the mold surface may be used with individual needles, flexible sheet needles, needle(s) on robotic arms, lasers, or other techniques. In another embodiment, the needle block may be used with a mold that has stupid holes that are non-perpendicular to the surface. Alternatively, the three-dimensional shape may be formed by a mold having multiple flat surfaces and a needle block that conforms to the flat surfaces. Similarly, molds having fool holes perpendicular to the mold surface may be used with individual pins, flexible sheet pins, pin(s) on a robot arm, or with limited lugs. However, other devices such as, but not limited to, push rods, punches, dies, or any sharps may be used to push through the perforations. In another embodiment, the pin block may be used with a mold that has stupid holes that are non-perpendicular to the surface. Alternatively, the three-dimensional shape can be formed by a mold having a plurality of flat surfaces and a pin block conforming to the flat surfaces.

The implant product may be removed from the mold and stop by manual force, or excess tissue may be separated from the stop and the product removed from the mold. Once the product is removed from the mold, it can be dried, lyophilized, or fully hydrated without the risk of delamination, or remain in the same hydrated state as when laminated. It may be. The product may be further manipulated to suit various medical applications. The product can be sterilized using standard techniques.

The mechanical properties of the product can be adjusted by adjusting the shape, by adjusting the number of layers, the sheets in layers, the type of sheets in layers, by adjusting the lug threading pattern, size, density, and shape. , ECM sheets from various animal and tissue sources, and by SPM sheet selection, can be tailored to medical application needs.

The open pores enhance the in vivo remodeling properties of the implant. The open pores are believed to promote ECM tissue contact with endogenous fluids and cells (by increasing the surface area of the implanted graft). The open pores also serve as conduits that allow extracellular fluid to pass through the implant. The open holes formed during the manufacture of the implant product of the present invention alleviate the accumulation of fluid between the sheets of implant fabrication by providing a conduit through which fluid can flow out of the tissue. become.

FIG. 5 shows an example of an instrument used to prepare the 3D tissue implant product of the present invention. FIG. 5A shows the mold 36 on the table 37. The mold 36 includes a plurality of fool holes 38 for receiving needles or pins. The fixing pin 39 is shown on the end of the mold 36 where the sheet of ECM or SPM will be fixed. FIG. 5B shows an alternative arrangement that uses multiple needles or pins. The mold 40 is shown on a stand 41. A sheet of ECM or SPM 42 is shown stretched onto the mold 40 and secured by attachment to a locking pin 43. The piercing needle block 44 (which may replace the push-through pin block) is attached to the block guide 45.

Delivery of Bioactive Materials The laminated implant products of the present invention may be used to deliver bioactive materials to an implant site. The bioactive material is a material that is either endogenous to the ECM used in the preparation of the implant product or that is incorporated into the ECM and/or polymeric material layer during or after the implant product manufacturing process. Good. Bioactive materials thus delivered to the implant site are known to be beneficial for promoting cellular functions, including wound healing and other desirable physiological and pharmacological functions.

Example 1: Base Laminated Product with 1 Rug Sheet and 4 Perforated Sheets ECM was prepared from vertebrate forestomach tissue according to the procedure described in US 8,415,159. Sheets of forestomach ECM were formed from segments of warm-blooded vertebrate forestomach tissue, including propria-submucosa.

Rug sheets were prepared by the following method: Four 5 mm diameter holes were punched into each corner of a 140 mm x 140 mm square of a sheet of lyophilized ECM using a cutting die and press. .. Place this sheet on a 140 mm x 140 mm flat acetal plate with a 5 mm diameter guide rod positioned equidistant from each corner to match the holes cut in the lyophilized ECM sheet and guide The lateral position of the ECM was fixed on the plate by a rod. The plate has a 29-row by 29-column grid of 3.1 mm diameter stupid holes located 3.5 mm center to center, providing a hole-free peripheral portion of about 20 mm width. This plate served as the mold. A retainer plate with guide holes for the lug cutters matching the pattern of fool holes was placed on top of the lyophilized ECM sheet. Stops were applied to secure the assemblies to each other. A spacer plate with a thickness of 5 mm having a stupid hole size and pattern matching the guide rod holes and stupid holes was placed on top of the holding plate. Guide rod holes and lug cutters for acetal blocks using lug cutting blocks that include acetal blocks with 2.5 mm diameter circular tube lug cutters spaced 3.5 mm in the center that match the stupid hole pattern. The guide holes were used to align the lug cutting blocks as the lug cutter was pushed through the ECM sheet. The assembly was placed in an arbor press and compressed. The rag cutting block was retracted, removed, rotated 180 degrees and repositioned. The assembly was placed in an arbor press and compressed. The lug cutting block was retracted and removed with the spacer. The rag cutting block was rotated 90° and replaced. The rug sheet thus formed, having a lug about 2.8 mm wide, was removed from the device.

Perforated sheets were prepared by the following method: Four overlapping sheets of fresh ECM were stretched onto a flat acetal plate with the same dimensions, holes and guide rods as the plate described above. The plate also had pins protruding from the sides used to secure the fresh stretched ECM sheet to the plate. A 5 mm diameter metal punch/guide was passed through a fresh ECM into each guide rod hole and the tissue pierced to secure the tissue in place. A 1 mm thick PTFE separation slip was placed on the fresh ECM. A retainer plate, which also had a grid of holes and a size and pattern that matched the grid of guide rods and stupid holes, was placed on top of the PTFE slip. Stops were applied to secure the assemblies to each other. A perforated needle block was used that included an acetal block with a size 11 embroidery machine needle placed in a grid with a pattern matching the stupid holes. The perforation block also had guide holes aligned with the guide rods that were used to align the perforated needle blocks on the mold with the needle toward the ECM sheet. The assembly was placed in an arbor press and compressed. The perforation block, stop plate, and PTFE separating slip were removed.

The laminated implant product was assembled by the following method: A rug sheet was placed on top of the perforated sheet and four guide holes were guided along the guide rods and aligned. The rug sheet was rehydrated with isotonic saline. The separation slip, hold down plate, and stop were replaced. A pin block was used that included acetal blocks with blunted pins arranged in a grid that matched the grid of stupid holes. The pin block also has guide holes aligned with the guide rods, with the pins facing the ECM sheet, the pin block lowered to the top of the retainer plate and aligned using the guide holes and guide rods. .. The assembly was placed in an arbor press and compressed. The pin block was removed, repositioned and compressed with an arbor press two more times. The assembly was disassembled. A scalpel was used to disconnect the device from the mold. Thus, a wet and flexible 100 mm x 100 mm interlocked laminated implant fabrication with the appearance of a thick, rough sheet was produced. The sheet was then lyophilized to produce a rigid multilayer device.

Example 2: Laminated product with 2 rug sheets and 3 perforated sheets A sheet of ECM was prepared as described in Example 1. Two rug sheets and three perforated sheets were similarly prepared as described in Example 1. Laminated products were assembled by the following method: Two lug sheets were placed on top of the perforated sheet and four guide holes were guided along the guide rods to align them side by side. The rug sheet was rehydrated with isotonic saline. The separation slip, hold down plate, and stop were replaced. A pin block was used that included acetal blocks with blunted pins arranged in a grid that matched the grid of stupid holes. The pin block also has guide holes aligned with the guide rods, with the pins facing the ECM sheet, the pin block lowered to the top of the retainer plate and aligned using the guide holes and guide rods. .. The assembly was placed in an arbor press and compressed. The pin block was removed, repositioned and compressed with an arbor press two more times. The assembly was disassembled. A scalpel was used to disconnect the device from the mold. Thus, a wet and flexible 100 mm x 100 mm laminated product with the appearance of a thick, rough sheet was produced.

Example 3: Laminated product with polypropylene rug sheet A sheet of ECM was prepared as described in Example 1. Rug sheets were prepared as described in Example 1, except that the sheets used were polypropylene mesh. The perforated sheet was prepared as described in Example 1. Laminated products were prepared by the following method: A polypropylene rug sheet was placed on top of the perforated sheet and four guide holes were guided along the guide rods and aligned. The rug sheet was rehydrated with isotonic saline. The separation slip, hold down plate, and stop were replaced. A pin block was used that included acetal blocks with blunted pins arranged in a grid that matched the grid of stupid holes. The pin block also has guide holes aligned with the guide rods, with the pins facing the ECM sheet, the pin block lowered to the top of the retainer plate and aligned using the guide holes and guide rods. .. The assembly was placed in an arbor press and compressed. The pin block was removed, repositioned and compressed with an arbor press two more times. The assembly was disassembled. A scalpel was used to disconnect the device from the mold. Thus, a wet and flexible 100 mm x 100 mm interlocked laminated implant product with the appearance of a thick, rough sheet was produced.

Example 4: Laminated Product with Different Sheets A sheet of Proprietary-Submucosal ECM was prepared as described in Example 1.
Lyophilized forestomach ECM rug sheets were prepared as described in Example 1. Four perforated sheets, one consisting of the small intestine proper-submucosa, one consisting of polypropylene mesh, one consisting of pericardium and one consisting of renal capsule matrix, were prepared according to the method of Example 1. Prepared. Laminated products were assembled by the following method: The rug sheet was placed on top of the perforated sheet and the four guide holes were guided along the guide rods and aligned. The rug sheet was rehydrated with isotonic saline. The separation slip, hold down plate, and stop were replaced. A pin block was used that included acetal blocks with blunted pins arranged in a grid that matched the grid of stupid holes. The pin block also has guide holes aligned with the guide rods, with the pins facing the ECM sheet, the pin block lowered to the top of the retainer plate and aligned using the guide holes and guide rods. .. The assembly was placed in an arbor press and compressed. The pin block was removed, repositioned and compressed with an arbor press two more times. The assembly was disassembled. A scalpel was used to disconnect the device from the mold. Thus, a wet and flexible 100 mm x 100 mm laminated product with the appearance of a thick, rough sheet was produced. Sheets of polypropylene were sandwiched within the product so as to provide the benefits of increased material strength and stiffness while not providing the appearance of the synthetic product and reducing exposure of the synthetic material at the tissue interface.

Example 5: Laminated product with rows of offset lugs A sheet of ECM was prepared as described in Example 1. The acetal plate used has centers of stupid holes arranged in a hexagonal grid, staggering each successive row of stupid holes to arrange the centers in a hexagonal grid, on a square dense array of straight columns. A lyophilized ECM rug sheet was prepared by the method of Example 1, except that it provided a denser stupa pattern. The resulting grid of holes consists of 33 rows repeating the inclusion of 28 or 29 stupid holes. The plate has a non-perforated peripheral portion about 20 mm wide. Four overlapping perforated sheets of fresh ECM were prepared by the method of Example 1. Laminated products were assembled by the following method: The rug sheet was placed on top of the perforated sheet and the four guide holes were guided along the guide rods and aligned. The rug sheet was rehydrated with isotonic saline. The separation slip, hold down plate, and stop were replaced. A pin block was used that included acetal blocks with blunted pins arranged in a grid that matched the grid of stupid holes. The pin block also has guide holes aligned with the guide rods, with the pins facing the ECM sheet, the pin block lowered to the top of the retainer plate and aligned using the guide holes and guide rods. .. The assembly was placed in an arbor press and compressed. The pin block was removed, repositioned and compressed with an arbor press two more times. The assembly was disassembled. A scalpel was used to disconnect the device from the mold. Thus, a wet and flexible 100 mm x 100 mm laminated product with the appearance of a thick, rough sheet was produced. Due to the offset row pattern of fool holes, the force required to perform lug cutting, drilling, and lug threading operations was reduced.

Example 6: Laminated Pseudoisotropic Product A sheet of ECM was prepared as described in Example 1. A lyophilized ECM rug sheet was prepared by the method of Example 1. Four perforated sheets of fresh ECM, except that the perforated sheets were arranged with collagen fiber orientations of approximately 36 degrees, -72 degrees, +72 degrees, and -36 degrees relative to the rug sheet placed at 0 degrees. Overlapping perforated sheets were prepared by the method of Example 1. The laminated pseudo-isotropic product was assembled by the following method: A rug sheet was placed on top of the perforated sheet and four guide holes were guided along the guide rods to align them side by side. The rug sheet was rehydrated with isotonic saline. The separation slip, hold down plate, and stop were replaced. A pin block was used that included acetal blocks with blunted pins arranged in a grid that matched the grid of stupid holes. The pin block also has guide holes aligned with the guide rods, with the pins facing the ECM sheet, the pin block lowered to the top of the retainer plate and aligned using the guide holes and guide rods. .. The assembly was placed in an arbor press and compressed. The pin block was removed, repositioned and compressed with an arbor press two more times. The assembly was disassembled. A scalpel was used to disconnect the device from the mold. Thus, a wet and flexible 100 mm x 100 mm laminated pseudo-isotropic product with the appearance of a thick, rough sheet was produced. Due to the different fiber orientations of the individual sheets, the product had the appearance of a pseudo-isotropic device with similar biomechanical properties in multiple plane directions.

Example 7: Laminated Product with Perforated Sheets Sandwiched Between Rug Sheets A sheet of ECM was prepared as described in Example 1. Two lyophilized ECM rug sheets were prepared by the method of Example 1. Before stretching the four overlapping sheets of fresh ECM onto the mold, one fresh rug sheet was placed except that one rug sheet was placed on the acetal plate at the 0 degree angle through the free ends of the lugs. Four overlapping perforated sheets of ECM were prepared by the method of Example 1. The result was a perforated sheet with four ECM layers stretched onto the mold with the lugs pressed into the stupa. The laminated sandwich product was assembled by the following method: a second lug sheet was placed on top of the perforated sheet through the free ends of the lugs at an angle of 180 degrees to the first lug sheet and four along the guide rods. The guide holes were guided and aligned. The rug sheet was rehydrated with saline. The separation slip, hold down plate, and stop were replaced. Arranged in a grid that matches the grid of the fool holes but offset 0.5 mm from the center of the fool holes and aligned to be positioned furthest away from the free end of the lug on the second lug sheet. , A pin block containing an acetal block with blunted pins was used. The pin block also has guide holes aligned with the guide rods, with the pins facing the ECM sheet, the pin block lowered to the top of the retainer plate and aligned using the guide holes and guide rods. .. The assembly was placed in an arbor press and compressed. The pin block was retracted, repositioned, and arbor pressed twice more. The assembly was removed from the arbor press and turned inside out.

Arranged in a grid that matches the grid of the fool holes, but offset 0.5 mm from the center of the fool holes and aligned to be positioned furthest away from the free end of the lug on the first lug sheet. A second pin block was used that included an acetal block with blunted pins. The pin block also had guide holes aligned with the guide rods, with the pins facing the ECM sheet, the pin block lowered to the top of the mold, and aligned using the guide holes and guide rods. The assembly was placed in an arbor press and compressed. The assembly was removed from the arbor press, the pin blocks on either side of the resulting fabrication were retracted and removed, the stops and holddown plates were removed, and the separation slips were removed. A scalpel was used to disconnect the device from the mold. Thus, a wet and flexible 100 mm x 100 mm laminated sandwich product with the appearance of a thick, rough sheet was produced.

Example 8: Laminated product with perforated sheets sandwiched between rug sheets, but with the rug hidden within the product A sheet of ECM was prepared as described in Example 1. 2. Two lyophilized ECM rug sheets, one according to the method of Example 1, one with a diameter of 5.0 mm centered, which results in a non-perforated peripheral portion about 21 mm wide. Prepared by the same method except the mold contained a 20 row by 20 column grid of 1 mm stupid holes. Before stretching the four overlapping sheets of fresh ECM onto the mold, one fresh rug sheet was placed except that one rug sheet was placed on the acetal plate at the 0 degree angle through the free ends of the lugs. Four overlapping perforated sheets of ECM were prepared by the method of Example 1. The result was a perforated sheet with four ECM layers stretched onto the mold with the lugs pressed into the stupa. A laminated sandwich product was assembled by the following method: A pin block was used that included an acetal block with a 20 row by 20 column array of blunted pins arranged in a grid that matched the grid of stupid holes. The pin block also had guide holes aligned with the guide rods, with the pins facing the ECM sheet and the pin block raised from underneath the mold and aligned using the guide holes and guide rods. The assembly was placed in an arbor press and compressed. The assembly was removed from the arbor press, the pin block retracted and removed, the stops and retainer plates removed, and the separation slips removed. The free ends of the lugs of the first lug sheet were flattened along the top of the perforated sheet in the direction that was initially oriented.

The second lug sheet was placed on top of the perforated sheet through the free ends of the lugs at an angle of 180 degrees to the first lug sheet, guiding the four guide holes along the guide rod and aligning them. .. The rug sheet was rehydrated with saline. The separate slip and hold down plate was replaced. The spacer block was placed on the holding plate and the fastener was replaced. A pin block was used that included an acetal block with a 20 row by 20 column array of blunted pins arranged in a grid that matched the grid of stupid holes. The pin block also has guide holes aligned with the guide rods, with the pins facing the ECM sheet, the pin block lowered to the top of the retainer plate and aligned using the guide holes and guide rods. .. The assembly was placed in an arbor press and compressed. The pin block was retracted and compressed with an arbor press two more times. The assembly was removed from the arbor press and disassembled. The fabrication was cut from the mold using a scalpel. Thus, a wet and flexible 100 mm x 100 mm mated laminated implant fabrication was produced with the appearance of a thick, rough sheet without protrusions.

Example 9: Hand laminated 3D product A sheet of ECM was prepared as described in Example 1. Rug sheets were prepared by the following method: 4 along one side 10 mm from the long end to the center, 1 short side to the center 10 mm, 111.5 mm, 130 mm, and 231.5 mm, 4 Is a relationship that closely resembles the first four holes along only the long side, with eight holes of 5 mm diameter in one sheet of lyophilized ECM, measuring 140 mm x 240 mm, Punching was done using a cutting die and press. This sheet is a 140 mm x 140 mm flat with 4 guide rods of 5 mm diameter equidistant from each corner to match the hole cut at the left end of the lyophilized ECM sheet. It was placed on the left side of a simple acetal plate and the position of the ECM was fixed on the plate by a guide rod. The plate has a 29-row by 29-column grid of 3.1 mm diameter stupid holes located 3.5 mm center to center, providing a hole-free peripheral portion of about 20 mm width. This plate served as the mold. A retainer plate with guide holes for the lug cutters matching the pattern of fool holes was placed on top of the lyophilized ECM sheet. Stops were applied to secure the assemblies to each other. A spacer plate with a thickness of 5 mm having a stupid hole size and pattern matching the guide rod holes and stupid holes was placed on top of the holding plate. Guide rod holes and lug cutters for acetal blocks using lug cutting blocks that include acetal blocks with 2.5 mm diameter circular tube lug cutters spaced 3.5 mm in the center that match the stupid hole pattern. The guide holes were used to align the lug cutting blocks as the lug cutter was pushed through the ECM sheet. The assembly was placed in an arbor press and compressed. The rag cutting block was retracted, removed, rotated 180 degrees and repositioned. The assembly was placed in an arbor press and compressed. The lug cutting block and spacer were removed. The rag cutting block was rotated 90° and replaced. The rug sheet thus formed, having a lug about 2.8 mm wide spanning a square of about 100 mm x 100 mm, was removed from the instrument and then repositioned so that the right hand side of the sheet was on the mold. The seat was lowered along the guide rods, the hold down plate was put in place, and the stops and spacer blocks were put in place to secure the assembly. The lug cutter block was lowered into place with the same orientation used when cutting the left hand side of the ECM sheet. The assembly was placed in an arbor press and compressed. The rag cutting block was retracted, removed, rotated 180 degrees and repositioned. The assembly was placed in an arbor press and compressed. The lug cutting block was retracted and removed with the spacer. The rag cutting block was rotated 90° and replaced. The rug sheet thus formed, having a lug about 2.8 mm wide spanning a rectangle of about 100 mm x 200 mm, was removed from the device.

A perforated sheet was prepared by the following method: A sheet of fresh ECM was drawn on a 140 mm diameter x 70 mm high hemispherical hollow acetal block dense with 2.0 mm holes perpendicular to the hemispherical surface. The ECM sheet was then positioned to take full advantage of any natural 3D morphology in possession. A sheet of ECM was held in the stretched position by an array of fixed pins on the underside of the block. This block had the function of a mold. Three more ECM sheets were applied, one by one, in the same manner.

A mated compliant laminated implant fabrication was assembled by the following method: A long rug sheet was partially hydrated and placed on top of an ECM wet sheet and stretched until it became flexible. .. Working along the row of lugs with two size 11 embroidery needles, lifting each lug and pushing the needle into the nearest available hole in the mold, making a perforation in four ECM layers, Was pushed through the perforation with a blunt end pin. Other rows in the area that needed to be rugged were treated in the same way. A scalpel was used to disconnect the device from the mold. The laminated implant product had a moist and flexible, thick, rough sheet appearance and conformed to the shape of the mold.

Example 10: 3D Single Point Laminated Product A sheet of ECM was prepared as described in Example 1. Rug sheets were prepared by the following method: 4 along one side 10 mm from the long end to the center, 1 short side to the center 10 mm, 111.5 mm, 130 mm, and 231.5 mm, 4 Is a relationship that closely resembles the first four holes along only the long side, with eight holes of 5 mm diameter in one sheet of lyophilized ECM, measuring 140 mm x 240 mm, Punching was done using a cutting die and press. This sheet is a 140 mm x 140 mm flat with 4 guide rods of 5 mm diameter equidistant from each corner to match the hole cut at the left end of the lyophilized ECM sheet. It was placed on the left side of a simple acetal plate and the position of the ECM was fixed on the plate by a guide rod. The plate has a 29-row by 29-column grid of 3.1 mm diameter stupid holes located 3.5 mm center to center, providing a hole-free peripheral portion of about 20 mm width. This plate served as the mold. A retainer plate with guide holes for the lug cutters matching the pattern of fool holes was placed on top of the lyophilized ECM sheet. Stops were applied to secure the assemblies to each other. A spacer plate with a thickness of 5 mm having a stupid hole size and pattern matching the guide rod holes and stupid holes was placed on top of the holding plate. Guide rod holes and lug cutters for acetal blocks using lug cutting blocks that include acetal blocks with 2.5 mm diameter circular tube lug cutters spaced 3.5 mm in the center that match the stupid hole pattern. The guide holes were used to align the lug cutting blocks as the lug cutter was pushed through the ECM sheet. The assembly was placed in an arbor press and compressed. The rag cutting block was retracted, removed, rotated 180 degrees and repositioned. The assembly was placed in an arbor press and compressed. The lug cutting block and spacer were removed. The rag cutting block was rotated 90° and replaced. The rug sheet thus formed, having a lug about 2.8 mm wide spanning a square of about 100 mm x 100 mm, was removed from the instrument and then repositioned so that the right hand side of the sheet was on the mold. The seat was lowered along the guide rods, the hold down plate was put in place, and the stops and spacer blocks were put in place to secure the assembly. The lug cutter block was lowered into place with the same orientation used when cutting the left hand side of the ECM sheet. The assembly was placed in an arbor press and compressed. The rag cutting block was retracted, removed, rotated 180 degrees and repositioned. The assembly was placed in an arbor press and compressed. The lug cutting block was retracted and removed with the spacer. The rag cutting block was rotated 90° and replaced. The rug sheet thus formed, having a lug about 2.8 mm wide spanning a rectangle of about 100 mm x 200 mm, was removed from the device.

A perforated sheet was prepared by the following method: A sheet of fresh ECM was packed with a pattern of 2.5 mm holes perpendicular to the hemispherical surface, showing an array of dense holes, 140 mm diameter x 70 mm height. Was stretched onto a hemispherical hollow acetal block of and the ECM sheet was positioned to take full advantage of any natural 3D morphology possessed. A sheet of ECM was held in the stretched position by an array of fixed pins on the underside of the block. This block had the function of a mold. Three more ECM sheets were applied, one by one, in the same manner. Sheets were punched using a robot arm.

The mated compliant laminated implant fabrication was assembled by the following method: A long rug sheet was partially hydrated and placed on top of an ECM wet sheet and stretched until it became flexible. , Aligned the lugs around the perimeter of the area that needs to be threaded over the drilled hole. A robotic arm was used to push each lug through each hole using a blunt pin instrument. A scalpel was used to disconnect the device from the mold. The laminated implant product had a moist and flexible, thick, rough sheet appearance and conformed to the shape of the mold.

Example 11: 3D multi-point laminated product.
Sheets of ECM were prepared as described in Example 1. Rug sheets were prepared by the following method: 4 along one side 10 mm from the long end to the center, 1 short side to the center 10 mm, 111.5 mm, 130 mm, and 231.5 mm, 4 Is a relationship that closely resembles the first four holes along only the long side, with eight holes of 5 mm diameter in one sheet of lyophilized ECM, measuring 140 mm x 240 mm, Punching was done using a cutting die and press. This sheet is a 140 mm x 140 mm flat with 4 guide rods of 5 mm diameter equidistant from each corner to match the hole cut at the left end of the lyophilized ECM sheet. It was placed on the left side of a simple acetal plate and the position of the ECM was fixed on the plate by a guide rod. The plate has a 29-row by 29-column grid of 3.1 mm diameter stupid holes located 3.5 mm center to center, providing a hole-free peripheral portion of about 20 mm width. This plate served as the mold. A retainer plate with guide holes for the lug cutters matching the pattern of fool holes was placed on top of the lyophilized ECM sheet. Stops were applied to secure the assemblies to each other. A spacer plate with a thickness of 5 mm having a stupid hole size and pattern matching the guide rod holes and stupid holes was placed on top of the holding plate. Guide rod holes and lug cutters for acetal blocks using lug cutting blocks that include acetal blocks with 2.5 mm diameter circular tube lug cutters spaced 3.5 mm in the center that match the stupid hole pattern. The guide holes were used to align the lug cutting blocks as the lug cutter was pushed through the ECM sheet. The assembly was placed in an arbor press and compressed. The rag cutting block was retracted, removed, rotated 180 degrees and repositioned. The assembly was placed in an arbor press and compressed. The lug cutting block and spacer were removed. The rag cutting block was rotated 90° and replaced. The rug sheet thus formed, having a lug about 2.8 mm wide spanning a square of about 100 mm x 100 mm, was removed from the instrument and then repositioned so that the right hand side of the sheet was on the mold. The seat was lowered along the guide rods, the hold down plate was put in place, and the stops and spacer blocks were put in place to secure the assembly. The lug cutter block was lowered into place with the same orientation used when cutting the left hand side of the ECM sheet. The assembly was placed in an arbor press and compressed. The rag cutting block was retracted and removed, rotated 180 degrees and repositioned. The assembly was placed in an arbor press and compressed. The lug cutting block was retracted and removed with the spacer. The rag cutting block was rotated 90° and replaced. The rug sheet thus formed, having a lug about 2.8 mm wide spanning a rectangle of about 100 mm x 200 mm, was removed from the device.

A perforated sheet was prepared by the following method: A sheet of fresh ECM was packed with a pattern of 140 mm diameter x 70 mm height hemispheres, clustered in a pattern with 2.5 mm holes parallel to each other showing an array of dense holes. The ECM sheet was oriented so that it stretched over a hollow cavity of the acetal block and maximized the benefits of any natural 3D morphology possessed. A sheet of ECM was held in the stretched position by an array of fixed pins on the underside of the block. This block had the function of a mold. Three more ECM sheets were applied, one by one, in the same manner. Sheets were punched using a robot arm. Needle block containing 56 size 11 embroidery needles at two positions, each indexing the mold on a table and resulting in an ECM that is perforated in a regular pattern on the lugged area. Was applied to the wet ECM.

The mated compliant laminated implant fabrication was assembled by the following method: A long rug sheet was partially hydrated and placed on top of an ECM wet sheet and stretched until it became flexible. , Aligned the lugs around the perimeter of the area that needs to be threaded over the drilled hole. 56 blunt ends arranged in the same pattern as the ones for the holes in the mold at two positions, with each index indexing the mold on the table and resulting in the ECM being lugged over the lugged area. A pin block containing pins was applied to the wet ECM. A scalpel was used to disconnect the device from the mold. The laminated implant product had a moist and flexible, thick, rough sheet appearance and conformed to the shape of the mold.

Example 12: Wet handling integrity test of rugged sheep forestomach matrix laminates and equivalent non-lugged laminates A rugged lyophilized laminate comprising one rug sheet and two perforated sheets, Prepared according to Example 1 and lyophilized. An equivalent non-lugged three-layer laminate was prepared by lamination according to the method of Floden et al. (8). Sheep forestomach matrix was prepared according to the procedure described in US 8,415,159. One layer of ovine forestomach wet matrix was placed on a flat surface with the abluminal surface facing up. The second layer was placed on top of the first layer, with the smooth abluminal surface facing down, in contact with the first layer. The third layer was placed on top of the second layer with the smooth abluminal surface facing upwards. Pressure was applied to remove any gaps between the layers. The three layer laminate was then freeze dried to create a three layer laminate.

Wet handling simulation tests were performed to compare the laminate strength of the lugged laminate and the equivalent non-lugged laminate. The lugged and non-lugged laminates were cut into 4 cm x 4 cm samples prior to wet handling testing. The final sample size for testing each of the laminates was 5 (n=5). Ten Petri dishes were each filled with 20 mL of 1×PBS. At T=0 time points, 4 cm x 4 cm laminates (rugged or unrugged) were introduced into Petri dishes and completely rehydrated in 1xPBS prior to wet handling testing. Upon rehydration, the laminate was picked at the corner with forceps and rocked for 5 seconds using left and right movement. The laminate was then reintroduced into PBS and folded in quarters. Then, while still folded, the stack was dropped into the lid of the Petri dish from a height of 20 cm. The laminate was then reintroduced into PBS and rocked for an additional 5 seconds. After the end of the test, the sample was evaluated for delamination. Delamination was defined as "more than 50% of any layer separated from the opposing layer". Samples delaminated by wet handling simulations were recorded as "fail". Wet handling simulation tests were performed at T=0 hours, T=12 hours and 24 hours. The laminate remained hydrated in PBS during the test. The results of the wet handling simulation test are shown in FIG.

Example 13: Wet handling simulation test of rugged sheep rumen laminate and equivalent non-lugged laminate A sheep rumen ECM was prepared as follows. Sheep rumen tissue was sourced from sheep under 1 year of age. The muscle layer was physically separated from the tissue and discarded. Residual tissue (approximately 1500 g) was incubated in 0.1% Triton TX-100 (10 L) with rocking for 4 hours at room temperature. The solution was discarded. Tissues were incubated for 18 hours at room temperature in a solution (10 L) containing 0.1% TX-100, 0.3% TRIS, and 0.15% EDTA. The solution was discarded. Finally, the tissue was rinsed three times with pure water (10 L) for 10 minutes with rocking at room temperature. Decellularized material was lyophilized as needed. A rug-threaded lyophilized laminate comprising one rug sheet and two perforated sheets was prepared according to Example 1 and lyophilized. An equivalent non-lugged three layer laminate was prepared according to Example 12. Wet handling simulation tests were performed according to Example 12 to compare the laminate strength of lugged laminates and equivalent non-lugged laminates. Results for the lugged and non-lugged sheep rumen ECM laminates are shown in FIG. The rugged sheep reticulum laminate did not remain intact after initial handling (T=0).

Example 14: Wet handling integrity test of lugged sheep pericardium laminate and non-lugged equivalent laminate Sheep pericardial ECM was prepared for lamination as follows. Sheep pericardium was supplied by sheep under 1 year of age. The pericardial sac was dissected from the heart and lungs and frozen to facilitate removal of fat from the tissue. Upon thawing, the tissue was washed and de-epithelialized according to Example 13. A lyophilized pericardium laminate containing one rug sheet and two perforated sheets was prepared according to Example 1 and lyophilized. An equivalent non-lugged three layer laminate was prepared according to Example 12. In particular, the pericardial shape resulted in a smaller device than the devices of Examples 1 and 12. Both laminates were cut into 2 cm x 2 cm laminates prior to wet handling testing. The final sample size for each laminate type was 4 (n=4). A wet handling simulation test was conducted according to Example 12. Results are shown in FIG.

While this invention has been described by way of example, it should be understood that variations and modifications can be made without departing from the scope of the invention as claimed. Further, where known equivalents exist to particular features, such equivalents are incorporated as if specifically referred to herein.

References

Claims (14)

A tissue implant product comprising two or more sheets of material, each sheet comprising an extracellular matrix (ECM) or polymeric material, the sheets interlocking a portion of one sheet with a portion of another sheet. By being laminated to each other,
The first sheet has a plurality of lugs, the second sheet has a plurality of holes, and each lug of the first sheet is placed through the holes of the second sheet, the first sheet Mesh with the second sheet ,
Each lug includes a portion of the sheet cut from the first sheet and remains connected to the sheet,
Tissue graft product. The tissue implant product of claim 1, wherein the holes and lugs are dimensioned such that the lugs engage the surface of the second sheet. Further comprising a third sheet positioned between the first sheet and the second sheet,
The third sheet has a plurality of holes aligned with the holes of the second sheet,
Lug of the first sheet, through which extends the third sheet of holes, the first sheet to the second sheet and third sheet engaged, any one of claims 1 or 2 The tissue graft product described in. Further comprising a third sheet having a plurality of lugs aligned with the holes of the second sheet,
The tissue implant according to any one of claims 1 or 2 , wherein the lugs of the third sheet extend through the holes of the second sheet and pass therethrough to mate the third sheet with the second sheet. One piece product. 5. The tissue graft fabrication of claim 4 , wherein each lug of the first sheet mates with a third sheet and each lug of the third sheet mates with the first sheet. Tissue according to claim 4 or 5 , wherein the third sheet comprises a plurality of lugs, the first sheet comprises a plurality of holes, the lugs of the first sheet being offset from the lugs of the third sheet. Graft product. Comprising at least one sheet ECM, tissue graft product according to any one of claims 1-6. The tissue implant fabrication of any one of claims 1-8, comprising a total of 3, 4, 5, 6, 7 , 8, 9, or 10 sheets of ECM and/or polymeric material. The tissue graft product of any one of claims 1 to 9 , wherein the ECM is formed from ruminant forestomach propria-submucosa. At least one sheet, polypropylene, polytetrafluoroethylene, polyglycolic acid, polylactic acid, comprising a synthetic polymeric material formed from Porigurekapuron -25, or polyester, any one of claims 1-9 The tissue graft product described in. The tissue graft product according to any one of claims 1 to 10 , wherein at least one sheet comprises a protein, a polysaccharide, a glycoprotein, a proteoglycan, or a glycosaminoglycan. The tissue implant product of any one of claims 1 to 12 , which is a substantially flat sheet or has a three-dimensional morphology shaped to fit where the product is to be implanted. By a portion engaged in one part of the sheet to another sheet, comprising the steps of laminating two or more sheet materials together, the tissue implant according to any one of claims 1 to 12 A method of preparing a product, wherein each sheet comprises an extracellular matrix (ECM) or polymeric material,
(I) placing a first sheet of material having a plurality of lugs on top of a second sheet having a plurality of holes;
(Ii) a step of pushing the lug of the first sheet through the hole of the second sheet, the hole and the lug engaging the first sheet with the surface of the second sheet to bring the first sheet into the second sheet. A process having dimensions such that it meshes with,
Including the method. 14. A method of preparing a tissue graft product according to claim 13 , wherein the ECM and/or polymeric material is a dry sheet.